Janus kinases (JAKs) are non-receptor tyrosine kinases that play an essential role in cytokine signaling (Darnell et al., Science 264: 1415-1421 (1994); Ihle, Adv. Immunol. 60: 1-35 (1995)). The JAK family consists of four evolutionary-conserved mammalian JAK proteins, JAK1, JAK2, JAK3 and TYK2, which are each approximately 120 kDa in molecular mass, and homologues in other vertebrates such as chicken, and zebrafish and drosophila. These kinases appear to be responsible for the transmission of signal by most cytokines and neurokines (Rane and Reddy, Oncogene 19: 5662-5679 (2000)). Accumulated evidence suggests that binding of cytokines to their receptors induces receptor oligomerization, which results in an increased affinity of the cytoplasmic domain of the receptor for the JAK kinases. As a consequence of this increased affinity, the JAK kinases are recruited to the receptors resulting in their phosphorylation and subsequent activation. The activated JAKs then phosphorylate the cytoplasmic tails of the receptors on target tyrosines residues, which in turn serve as the docking sites for the Src-homology-2 (SH2) domains of signal transducer and activation of transcription (STAT) proteins. The recruited STATs are phosphorylated by JAKs on specific tyrosine residues, which causes their release from the receptor and finally dimerization through a reciprocal phosphotyrosine-SH2 domain interaction (Chen et al., Cell 93:827-839 (1998); Becker et al., Nature 394: 145-151 (1998)). The dimerized STAT proteins then translocate to the nucleus where they act as transcription factors.
A unique feature of the domain-structure of JAKs that distinguishes them from other tyrosine kinases is the presence of two tandem domains with extensive homology to tyrosine kinases, a C-terminal catalytic domain and an immediately preceded pseudokinase domain (Ihle, supra). The pseudokinase domain lacks canonical residues that are essential for catalytic function. Several lines of evidence suggest that this domain regulates catalytic activity and autophosphorylation (Saharinen et al., Mol. Biol. Cell 14: 1448-1459 (2003); Saharinen et al., Mol. Cell. Biol. 20: 3387-3395 (2000); Saharinen et al., J. Biol. Chem. 277: 47954-47963 (2002); Chen et al., Mol. Cell. Biol. 20: 947-956 (2000)).
In addition to the two kinase domains, JAKs contain an N-terminal band four-point-one, erzin, radixin, moesin (FERM) homology domain and an SH2-like domain (Girault et al., Trends Biochem. Sci. 24: 54-57 (1999)). The FERM domain is a 300-amino acid protein-protein interaction module that mediates receptor interactions and is important for the preservation of proper catalytic function (Terawaki et al., Acta Crystallog. D59: 177-179 (2003); Smith et al., J. Biol. Chem. 278: 4949-4956 (2003); Hamada et al., EMBO J. 19: 4449-4462 (2000); Hamada et al., EMBO J. 22: 502-514 (2003); Pearson et al., Cell 101: 259-270 (2000); Zhou et al., Mol. Cell. 8: 959-969 (2001)).
The activity of JAKs is also regulated by the two tyrosines in the activation loop of the catalytic domain (Gauzzi et al., J. Biol. Chem. 271: 20494-20500 (1996); Feng et al., Mol. Cell. Biol. 17: 2497-2501 (1997); Zhou et al., Proc. Natl. Acad. Sci. USA 94: 13850-13855 (1997)). In JAK3, phosphorylation of Tyr980 and Tyr981 results in positive and negative regulation of its enzymatic activity, respectively (Zhou, supra).
JAK3 is predominantly expressed in lymphoid and myeloid cell lines and in hematopoietic tissues such as the thymus, bone marrow, spleen, and fetal liver (Rane and Reddy, Oncogene 21:3334-3358 (2002)). In contrast, other JAKs are ubiquitously expressed. JAK3 specifically associates with the common γ chain (γc) of the cytokine receptors for interleukin-2 (IL-2), IL-4, IL-7, IL-9, IL-15 and IL-21 (Kisseleva et al., Gene 285:1-24 (2002); O'Shea et al., Cell 109 Suppl: S121-131 (2002)). In humans, mutations in JAK3 or γc result in severe combined immunodeficiency (SCID), which is characterized by the absence of circulating mature T cells and natural killer cells, but not B cells (T−B+SCID) (Notarangelo et al., Hum. Mutat. 18: 255-263 (2001); Roberts et al., Blood 103:2009-2018 (2004); Epub in November 2003). JAK3−/− mice also exhibit severe immunodeficiency (Thomis et al., Science 270: 794-797 (1995)).
Therapeutic targeting of JAK3 kinase has received particular attention, because the effects owing to the complete absence of JAK3 are limited to the immune system. Several JAK3 inhibitors, such as JANEX-1, AG-490, WHI-P154 and PNU156804 have been reported (Sudbeck et al., Clin. Cancer Res. 5: 1569-1582 (1999); Cetkovic-Cvrlje et al., Arzneimittelforschung 53: 648-654 (2003); Cetkovic-Cvrlje et al., Clin. Immunol. 106: 213-225 (2003); Saemann et al., Transplantation 75: 1864-1874 (2003); Stepkowski et al., Blood 99: 680-689 (2002)). More recently, Pfizer reported an orally active JAK3 selective inhibitor, CP-690,550 as an immunosuppressive agent in mouse and monkey transplant models (Changelian et al., Science 302: 875-878 (2003)). Collectively these data suggest that JAK3 is an attractive pharmacologic target for the treatment of immune-mediated transplant rejection (Kirken, Transplant Proc. 33: 3268-3270 (2001)).
Despite its importance in SCID and as a clinical target for immunosuppression, very little is known about the three-dimensional structure of JAK3. Drug design for human therapy has been hampered because the structure of JAK3 was not previously known. Without structural information of JAK3, the detailed knowledge of the mechanism is limited and progress of designing drugs as specific inhibitors is impeded. Structural information on the unique features of the active site of human JAK3 would facilitate drug discovery.